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Conclusions: Coadministration of tetracycline HCl with the contraceptive patch did not affect the pharmacokinetics of 17d-NGM or EE. Based on these data, tetracycline is not expected to affect the contraceptive efficacy of EVRA”. Treatment with EVRA”, with or without tetracycline HCl, was safe and well tolerated.

FC1.23 DAILY FEATURED STUDIES I

FC1.23.01 EXPOSURE OF PRIMARY HUMAN TROPHOBLAST CELLS TO CYTOKINES INDUCES SURFACE AND SOLUBLE CHEMOKINE RECEPTORS (CCRs) S. Vassiliadis (l), E. Koumantakis (2), I. Athanassakis (1). (1) Dept. Biology, University of Crete, Heraklion, Greece. (2) Dept. OBIGYN, University Hospital, Heraklion, Greece.

Objectives: We examined the ability of IFN71 and IL-4 to induce CCR3 or CXCR4 on mature human trophoblast cells from term placenta since expression of CCRs on trophoblast cells may provide a valid mechanism for the in utero transmission of HIV. Study Methods: We defined the constitutive and inducible expression of surface CCR3 and CXCR4 on primary human trophoblasts during short periods of cell culture by immunofluorescence after exposure to IFN-71 and IL-4. In addition, released CCR activity was also monitored by ELISA. Results: Kinetic experiments show that the constitutive expression of both CCR3 and CXCR4 reach a peak of expression after 6 hours of culture, whereas by 24 hours they have almost disappeared. In the presence of IFN-71, CCR3 increases in expression after 4 hours of incubation, reaching highest levels at 24 hours of culture, whereas CXCR4 is kept at lower levels as compared to non-treated cells. In the presence of IL-4, CCR3 declines from 2 to 8 hours of culture to increase again at 24 hours by 50%. Under the IL-4 stimulus, CXCR4 shows a peak of expression at 8 hours of culture. Interestingly, we detect soluble CCR activity in the culture supernatants of trophoblast cells, which follows an inversely proportional pattern of that corresponding to surface expression. Conclusions: Surface induced expression of CCRs on trophoblast cells has a fast turnover rate between 2 and 8 hours of culture. The released CCR activity, however, may account for the development of an inhibitory mechanism against viral transmission by absorbing virions in the extracellular matrix.

FC1.23.02 RECURRENT VENOUS THROMBOEMBOLISM DURING HORMONE REPLACEMENT THERAPY. RESULTS OF THE ESTROGEN IN VENOUS THROMBOEMBOLISM TRIAL E. Ovintad, E. Hoibraaten, H. Arnesen, S. Larsen, E. Wickstrom, PM Sandset, Ullevaal University Hospital, dept of Hematology, Oslo, Norway.

Objectives: The aim of the study was to determine if hormone replacement therapy (HRT) alters the risk of venous thromboembolism (VTE) in high risk women; i.e. women with previous VTE. Study Methods: Randomized, double-blinded, and placebo-controlled clinical trial with a stratified design combined with a double-triangular sequential design. 140 females with previously verified VTE, were randomized to either 2 mg estradiol plus lmg norethisteron acetate one tablet daily (n=71), or placebo (n=60). The primary outcome was the occurrence of deep venous thrombosis or pulmonary embolism. Results: 8 women in the hormone group and one woman in the placebo group experienced VTE. The study was terminated early based on the results of circumstantial evidence emerging during the trial. The final analysis gave a median unbiased estimate of the incidence of VTE of 2.2% and 10.6% in the placebo group and HRT group respectively (p=O.O4). In the hormone group, four women experienced deep venous thrombosis, three pulmonary embolism and one cerebral sinus vein thrombosis. All these events happened within 220 days after inclusion. The one patient in the placebo group suffered pulmonary embolism at day 413. Conclusions: In women with previous VTE, HRT increases the risk of recurrent VTE and should be avoided. This study confirms the result of the epidemiological studies showing a small increased risk of VTE on HRT, which might be generalized to most women

FC1.23.03 DEVELOPMENT-DEPENDENT GLUCOCORTICOID METABOLISM IN HUMAN OVARIAN GRANULOSA CELLS P.Y.K.Yon& J.Thong, ‘R.Andrew, ‘B.Walker, S.G.Hillier; University of Edinburgh, Centre For Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, ‘Molecular Medicine Centre, Crewe Road, Edinburgh EH4 2XU, United Kingdom

Objectives: llfl hydroxysteroid dehydrogenase (llflHSD), which is responsible for the bidirectional conversion between cortisol (F) and its inactive form, cortisone (E), has been implicated in the regulation of ovarian folliculogenesis and oogenesis. Expression of its two known isomers (llflHSD1, llflHSD2) by human granulosa cells (GC) has been shown to be developmentally regulated. Immature GC (IGC) almost exclusively express llgHSD2 mRNA while luteinised GC (LGC) express predominantly llflHSD1 mRNA. The aims of this study were: (1) to confirm the switch in 1lgHSD expression from type 2 to type 1 by measuring the interconversion between E and F in IGC and LGC, and follicular fluid levels of E and F, and (2) to determine if 1 lgHSD1 reductase activity (E to F conversion) is gonadotrophically regulated in vitro. Methods: LGC were aspirated from periovulatory follicles in IVF patients during oocyte recovery, 35h after injection of hCG. IGC were collected from antral follicles (3.16mm) in women undergoing oophorectomy for benign gynaecological conditions. Cells (l-5 x 10’) were incubated for 4h at 37°C $n medium 199 containing 50pmol of E and F, which included O.lpCi [ HI-E or F, followed by steroid extraction, thin-layer chromatography, and spectrometry to determine % conversion between E and F. For objective (2), IGC was incubated with FSH and LH for 48h before the 4h incubation described above. Experiments in triplicate. Results: Results are mean&EM values. % conversion of E to F by LGC (n=7) was 36.3*3.7%, compared with only 0.6*0.4% for IGC (n=5) (p<O.Ol). Conversion of F to E was similar for both groups: 31.0*2.9% for LGC and 41.7*7.4% for IGC. In IGC cultures (n=3), FSH (long/ml) increased conversion of E to F by a factor of 57.8e19.9 times over control incubations, without added gonadotrophins, while the corresponding value for LH (long/ml) was 3.59e2.11. The FSH effect was dose-dependent. The ratio of follicular fluid levels of F : E was almost 3 times greater in periovulatory than immature follicles (p<O.Ol). Conclusion: These data provide experimental confirmation of a switch in 1lgHSD expression from type 2 in immature follicles to type 1 in periovulatory follicles. We have shown for the first time that llflHSD1 reductase activity is upregulated in vitro by FSH and to a much lesser extent LH. The significance of increased follicular cortisol formation around the time of ovulation remains unclear, but is consistent with an anti-inflammatory role at follicular rupture to mediate repair and healing afterwards.

FC1.23.04 DIFFERENTIALLY EXPRESSED GENES IN NORMAL HUMAN MAMMARY TISSUE DURING TAMOXlFEN THERAPY (LUD 5646). I. L. Gebrim, Federal University SBo Paula Brazil, Rua Pedro de Toledo 781, 4 th floor, SBo Paula, Brazil, 04039032.

Introduction: Tamoxifen (TAM) an orally effective, synthetic antiestrogen used first in women with metastatic breast cancer disease has become an essential part of any therapeutic strategy for the control and prevention of breast cancer. In order to identify TAM responsive genes we performed cDNA arrays using total RNA isolated from normal breast tissue from patients treated with placebo or TAM (20 mgiday) for 4 weeks. The cDNA fragments used on membranes were generated by the Human Cancer Genome Project, funded by the Ludwig Institute for Cancer Research and FAPESP (Fonda@0 de Amparo ?I Pesquisa do Estado de SBo Paula, BRAZIL). This is a program for human gene discovery and human coding region compilation based on a novel concept for the high throughput sequencing of human open reading frames. The project consists entirely on the sequencing and analysis of short cDNA fragments generated preferentially from the central coding portions of expressed human genes obtained from tumors including breast, ovarian and cervical cancer. The fragments are termed ORESTES (Open Reading frame ESTs) and are generated using a strategy developed and patented by the Ludwig Institute. Within the project it is aimed to assemble the data, together with existing data in GenBank and

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Unigene, into annotated potential complete open reading frames (pORFs). Objectives: To isolate up or down regulated genes induced by short term TAM therapy. Patients and Methods: Human breast normal tissue were obtained at the Mastology Group, Gynecology Department at the Federal University of SBo Paula Brazil. This randomized, double-blind study included 20 women with fibroadenoma, half of them (10 women) received 20 mg of TAM/day during 30 days and the other half received placebo for the same time. During lumpectomy, with local anesthesia, a fragment of adjacent normal breast tissue were collected and immediately frozen in liquid nitrogen. Total RNA were isolated from tissues using Trizol (Life Technologies) and then used as template for reverse transcriptase reaction (Superscript II, Life Technologies) in the presence of [32P] dCTP. Membranes containing 96 different cDNA fragments were assembled using clones generated by the Human Cancer Genome Project (FAPESP-Ludwig Institute for Cancer Research) in SBo Paula, Brazil and hybridized overnight to the probes prepared before. Results: Three differentially expressed genes were found down- regulated during TAM treatment in the pool of RNA from TAM treated women. The isolated and down-regulated genes were Insulin-like Growth Factor Binding Protein 3, Calmodulin-dependent Protein Kinase II and Tissue Inhibitor of Metalloprotease 3. These results suggests that TAM might exerts its actions, on normal human breast tissue, through mechanisms that include Insulin-like Growth Factors and invasion enzymes. In addition, the results suggest that there might be diverse effects of hormones and neurotransmitters that utilize Ca2+ as a second messenger.

FC1.23.05 GENE TRANSFER TO PLACENTA Mikko 0. Hiltonen’, Annaleena HeikkilP ‘J Mikko P. Turunen’, Seppo Heinoner?, Mari Niemi’, Heikki RBsPner?, Tuomas Rissanen’, Veli- Matti Kosma~, Anna-Mari Turunen’, Leea Keski-NisulaZ, Fuju Chang4 and Seppo YIP-Herttualal,6,7 ‘A.I. Virtanen Institute, ‘Department of Gynecology and Obstetrics, 3Department of Radiology, “Department of Pathology, 5Department of Pathology and Forensic Medicine, 6Department of Medicine and ‘Gene Therapy Unit, University of Kuopio, Kuopio, Finland

Objective: If gene therapy could correct placental functional deficiencies in pre-eclampsia, both maternal and fetal morbidity from the disease could be markedly reduced. This study was undertaken to investigate whether adenovirus mediated gene transfer is feasible to placenta in a rabbit model. Study methods: Ten New Zealand White rabbits were anesthetized on day 21 of gestation. Adenovirus vector carrying the beta-galactosidase gene run by CMV promoter was injected via a catheter to both uterine arteries under angiographic control. The animals were sacrificed on day 24 of gestation and detection of vector was carried out by PCR and X- gal histochemistry. Results: Intravascular gene transfer produced high expression of enzyme-encoding trangene in placenta demonstrated by beta- galactosidase activity in the nuclei of placental cells and by PCR on extracts of the trophoblastic tissue. X-gal histochemistry revealed no leak to the fetal compartment and structurally normal fetuses were viable at the time of termination. Accordingly, gross light-microscopic histology of the fetoplacental entity was entirely normal. Conclusion: Gene transfer produces high-level transgene expression in placenta without marked leakage to fetal tissues and may become a new method to treat placental disorders.

FC1.23.06 FIGmING FEMALE FOETICIDE IN INDIA: LEGALLY AND ALSO PROFESSIONALLY A. Basu-Senguuta (l), K. Banik (2) (1) Bengal Obstetrics and Gynecology Society, Calcutta, India. (2) Indian Medical Association, Calcutta, India.

The Constitution of India guarantees the fundamental rights of its citizens including non-discrimination on the basis of gender. That violation is taking place through abortion of female fetuses in very large numbers, which is evident from the declining female male ratio in this country. This is simply due to gender bias as boys are preferred to girls. But this preference is taking a very sinister form. Thousands of medical

clinics have sprung up in some states for the determination of sex and termination of pregnancy, if possible, immediately. Abortion was illegal in India, but in 1971, with an eye to population control, the government introduced the Medical Termination of Pregnancy Act that made abortion legal if recommended by a physician and it was found that the number of abortions soared in the seventies. When the equipment for sex determination tests became available in the eighties, the termination of female fetuses increased alarmingly. The heinous practice finally compelled the government to introduce a specific act. The Prenatal Diagnosis Techniques (Regulation and Prevention of Misuse) Act, 1994, made both USG detection of sex and communication of the sex of the fetus in any manner to the mother and family a punishable offence. However, no case has been registered yet because the family is always in league with the physicians. Failing to contain the menace through legal instruments, professional bodies that would generally stand by the physicians are being mobilized. Indian Medical Association, the largest body of the physicians in the country has come forward to make India free from female feticide. The members have taken a pledge that they will not indulge themselves or be a party to the crime. IMA jointly with UNICEF has worked out an action plan, including a countrywide study on the social, legal and medical issues. The authors, leading state level leaders in the movement, report on the developments in recent years on the legal and professional side in stopping the unethical practice of female feticide.

FC1.23.07 OVARIAN CANCER FOLLOWING INFERTILITY AND FERTILITY DRUG USE. A DANISH COHORT STUDY OF MORE THAN 54,000 INFERTILE WOMEN S. Kriiner Kiaer I, H. Sharif I, .I. Bock ‘, .I. H. Olsen ’ ’ Institute of Cancer Epidemiology, Danish Cancer Society, Copenhagen, Denmark ’ Dept. OBIGYN, Rigshospitalet, Copenhagen, Denmark

Objectives: The overall aim of the study was to examine the cancer occurrence in infertile women. The specific aims of this presentation are: 1) to compare the ovarian cancer incidence in a cohort of infertile women with that of the general population, 2) to elucidate whether infertile women treated with fertility drugs are at a higher risk of ovarian cancer/borderline tumour than infertile women who never received these medications. Study methods: We have established a cohort of women evaluated for infertility problems at hospitals and private fertility clinics in Denmark 1960-1997. Relevant patients were identified from medical files, microfilms, and local computerised registration systems. Further, we identified patients from the Danish National Registry of Patients, containing more than 99% of disharges since 1977. The cohort was linked to the Medical Birth Register to obtain information on e.g. lifetime number of pregnancies. Cancer occurrence was ascertained by linking the cohort to the Danish Cancer Registry, comprising almost all cancers diagnosed in Denmark since 1943. From hospitaVclinic files of all the identified cancer cases and from a random sample (n-1300) of the cohort, we abstracted detailed information on infertility treatment. Results: In all, 54,379 women with an infertility diagnosis were included in the study. A total of 1086 women developed cancer during follow-up, including 76 ovarian cancers and 42 ovarian borderline turnours. Conclusion: We have established one of the largest cohorts of infertile women. Due to the existence of nation-wide registers in Denmark and to the fact that all Danish inhabitants have a unique personal ID-number, it is possible to do follow-up studies in Denmark with a nearly complete follow-up. Age-standardized incidence ratios (SIR) as well as results from the on-going case-cohort analyses regarding the role of fertility drug use will be presented.

FC1.23.08 PERMEABILITY OF FETAL MEMBRANES TO CATIONS IN PRETERM LABOR. T.Laudanski cl), A.Lemancewicz (l), H.Laudanska (l), S.Batra (2), (1) Medical University of Bialystok, M. Curie Sklodowskiej 24a, Bialystok, Poland, 15276, (2) University of Lund, Lund, Sweden.

Objective: It is recognized that fetal membranes may play a central role in paracrine signaling during the initiation of parturition. Since no